Investigation of salt tolerance in cotton germplasm by analyzing agro-physiological traits and ERF genes expression

The development of genotypes that can tolerate high levels of salt is crucial for the efficient use of salt-affected land and for enhancing crop productivity worldwide. Therefore, incorporating salinity tolerance is a critical trait that crops must possess. Salt resistance is a complex character, controlled by multiple genes both physiologically and genetically. To examine the genetic foundation of salt tolerance, we assessed 16 F1 hybrids and their eight parental lines under normal and salt stress (15 dS/m) conditions. Under salt stress conditions significant reduction was observed for plant height (PH), bolls/plant (NBP), boll weight (BW), seed cotton yield (SCY), lint% (LP), fiber length (FL), fiber strength (FS), potassium to sodium ratio (K+/Na+), potassium contents (K+), total soluble proteins (TSP), carotenoids (Car) and chlorophyll contents. Furthermore, the mean values for hydrogen peroxide (H2O2), sodium contents (Na+), catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and fiber fineness (FF) were increased under salt stress. Moderate to high heritability and genetic advancement was observed for NBP, BW, LP, SCY, K+/Na+, SOD, CAT, POD, Car, TSP, FL, and FS. Mean performance and multivariate analysis of 24 cotton genotypes based on various agro-physiological and biochemical parameters suggested that the genotypes FBS-Falcon, Barani-333, JSQ-White Hold, Ghauri, along with crosses FBS-FALCON × JSQ-White Hold, FBG-222 × FBG-333, FBG-222 × Barani-222, and Barani-333 × FBG-333 achieved the maximum values for K+/Na+, K+, TSP, POD, Chlb, CAT, Car, LP, FS, FL, PH, NBP, BW, and SCY under salt stress and declared as salt resistant genotypes. The above-mentioned genotypes also showed relatively higher expression levels of Ghi-ERF-2D.6 and Ghi-ERF-7A.6 at 15 dS/m and proved the role of these ERF genes in salt tolerance in cotton. These findings suggest that these genotypes have the potential for the development of salt-tolerant cotton varieties with desirable fiber quality traits.


Materials and methods
The experimental work was carried out on the research premises of FB Genetics, Four Brothers Group, Pakistan during the cropping year 2021.The experimental site is located at 74˚ east longitude and 31˚ north latitude.Under normal field conditions, the F 0 seeds of eight genotypes were sown and then, in a Line × Tester manner, were crossed.Four genotypes (namely FBS-FALCON, FBS-SMART, FBG-222 and Barani-333) were used as lines and four genotypes (namely JSQ-White Hold, FBG-333, Barani-222 and Ghauri-3) were used as testers (Table 1).When the cotton genotypes were flowering, they were crossed in a line × tester way (4 × 4).To create selfed seeds, certain buds were also wrapped in glassine bags.The mature selfed and crossed bolls were harvested and preserved.Subsequently, the cotton fibers from each group were processed separately using a single roller ginning machine (Testex, Model: TB510C) after being picked.In the next growing season, 24 genotypes (including 8 parents and their 16F1 hybrids) were grown in containers with normal soil (1.9 dS/m) under RCBD with three replications.After the emergence of seedlings, healthy specimens were selected for each genotype in every replication, all of which were subsequently subjected to salt stress through irrigation water with a known EC.The Analysis of soil properties of the experimental site was also conducted (Table 2).The salt stress was induced following a well-established procedure 8 .This method involves gradually increasing the salinity to a target level of ~ 15 dS/m in two distinct phases to avert seedling injury and ensure survival.Sodium chloride (NaCl) was calculated and added to the groundwater using the U.S. Salinity Lab formula 19 .

Amount of sodium chloride g/kg =
Equiv.Wt of NaCl × Saturation Percentage 100 × 1000 × TSS Table 1.Material used in breeding study.In this equation, TSS (Total Soluble Salts) represents the difference in EC (desired EC-initial EC) multiplied by 12.66, and the saturation percentage of the soil was established at 42%.
During the initial phase, the 2-week-old seedlings were subjected to a salinity level of 7.5 dS/m, which necessitated the addition of approximately 17.74 g of NaCl to each 10 kg pot.Subsequently, in the second phase, the 4-week-old seedlings were subjected to an escalated salinity level of 15 dS/m, which required a further addition of about 23.72 g of NaCl per 10 kg pot.Thus, approximately 41.46 g (17.74 g + 23.72 g) of NaCl was added per 10 kg pot in the two phases to attain the desired salinity level.After reaching this level, no further salt was added.Following this precise modulation of salinity, standard agricultural procedures were then followed for irrigation.This careful approach enabled us to achieve gradual increments in salinity while preventing seedling injury and ensuring survival throughout the experiment.
All agronomic procedures were appropriately carried out from the time of sowing until harvesting.Figure S1 displays the environmental conditions, including temperature, humidity, and rainfall, during crop growth.Upon reaching maturity (i.e., after 140 days of growth and > 80% completion of boll formation), the following parameters were measured from five chosen plants from each replication of both normal and saline treatment: plant height, bolls/plant, boll weight, seed cotton yield/plant, lint%, H 2 O 2 , SOD, POD, CAT, TSP, chlorophyll contents (a & b), carotenoids, fiber strength, fiber length, fiber fineness, Na + , K + and K + /Na + ratio.

Ion analysis
The analysis of sodium and potassium ions involved grinding hot air-dried leaves using a pestle and mortar.Concentrated nitric acid and sulfuric acid in a 2:1 ratio were used for the digestion of ground leaves on the hot plate.Then digested samples were cooled down by adding distilled water at room temperature.A flame photometer (410 Flame Photometer) was used to take out readings and then the K + /Na + ratio was calculated.

Hydrogen peroxide (µmol/g-FW)
Bernt and Bergmeyer 20 method was used to measure the hydrogen peroxide of treated and control samples.0.5 g of leaf sample was homogenized using liquid nitrogen for each control and treated group.1.5 ml of 100 mM potassium phosphate buffer (pH 6.8) was used for the suspension of ground leaves then the suspension was centrifuged at 18,000×g for 20 min at 40 °C.The supernatant (0.25 ml) was taken out and mixed with 1.25 ml of peroxidase reagent containing; 40 μg peroxidase/ml, 0.005% (w/v) O-dianizidine and 83 mM potassium phosphate buffer (pH 7.0) at 30 °C to initiate the reaction.After 10 min, the reaction was halted by adding 0.25 ml of 1 N perchloric acid and centrifuging the mixture at 5000×g for 5 min.A spectrophotometer (NanoDrop™ 8000 Spectrophotometer Thermo Fisher Scientific, Sweden) was used to measure the absorbance at 436 nm and the amount of H 2 O 2 was determined based on an extinction coefficient of 39.4 mM −1 /cm 21 .The SOD activity was measured in terms of enzyme units that inhibited the photochemical reduction of nitro blue tetrazolium (NBT).

Superoxide dismutase
To quantify SOD, a reaction mixture containing 100 μl of enzyme extract, potassium phosphate buffer (pH 5), 200 μl of methionine, 200 μl of Triton X, 100 μl of NBT, and 800 μl of distilled water was prepared.The mixture was exposed to ultraviolet light for 15 min, followed by the addition of 100 μl of Riboflavin.The absorbance at 560 nm was measured using a spectrophotometer.

Peroxidase (U/mg protein)
The same enzyme extracted for measuring SOD was used to measure POD values.It is calculated as the number of enzyme units that oxidized guaiacol.The reaction mixture was prepared by mixing 100 μl H 2 O 2 (40 mM), 100 μl guaiacol (20 mM) with 100 μl of enzyme extract, and 800 μl potassium phosphate buffer (pH 5) in Eppendorf tube and a spectrophotometer was used to measure absorbance at 470 nm wavelength 22 .

Total Soluble Proteins (mg/g-FW)
To determine the Total Soluble Proteins (TSP), leaf tissue extraction was carried out using vortex and centrifugation in a phosphate buffer (pH 4).Furthermore, a 40 μl portion of the same enzyme extract was mixed with 160 μl of Bradford reagent, added to an ELISA plate, and subjected to spectrophotometer readings at a wavelength of 595 nm absorbance 23 .

Chlorophyll contents and carotenoids assay
The chlorophyll and carotenoid contents were measured by following 24 .0.5 g of cotton leaf sample underwent crushing in 8-10 ml of 80% acetone (volume/volume).Subsequently, homogenization occurred through filter paper, and the absorbance of the resulting solution was measured at wavelengths of 645 and 663 nm.The quantification of chlorophyll a, chlorophyll b, and carotenoids followed this procedure.The chlorophyll a & b and carotenoids were estimated as follows, where, W = weight of leaf sample, V = volume of sample, Em = 2500.

qRT-PCR of Ghi-ERF-2D.6 and Ghi-ERF-7A.6 in tolerant cotton genotypes
The newly emerged leaves of control and stressed (15 dS/m) genotypes at two leaves stage were sampled.The RNA was extracted using a kit protocol (RNAprep Pure Plant Kit by Tiangen, Beijing, China) and the quality and integrity of the RNA were checked using gel electrophoresis and NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) respectively.The cDNA was prepared using (PrimeScript ® RT Reagent Kit, Takara Biotechnology Co., China).Three repeats of both genes were used technically.The real-time differential expression of mRNA of both genes was measured using qRT-PCR (Maxima SYBR Green)/ROX qPCR Master mix (2X), cat#K0221, Thermo scientific, USA).Gene normalization was obtained using GAPDH as an internal control.The genespecific primers were used for the expression of both genes (Table 3).In a PCR reaction gene-specific short-length primers for ERF2 and ERF7 gene were used.A total volume of PCR reaction mixture was prepared in a volume of 25 µl.General PCR conditions using short-length primers were as follows in this study: firstly, one cycle of initial denaturation at 95 °C for 3 min; secondly, 28 cycles of denaturation at 95 °C for 45 s, annealing at 66 °C for 45 s, and extension at 72 °C for 2 min; finally, one cycle of final extension at 72 °C for 10 min.

Fiber quality parameters
A seed cotton sample was weighed and subjected to a single roller ginning machine, to separate lint from the seeds.The percentage (%) of the lint was then calculated.This was achieved by dividing the weight of the lint by the weight of the seed cotton in the sample, the obtained value was then finally expressed as a percentage.Furthermore, employing a high-volume instrument (HVI-900, USTER, USA), the resulting lint was given an in-depth analysis to determine characteristics such as fiber strength, fiber fineness and length parameters.

Statistical analysis
The split-plot analysis of variance (ANOVA) was performed with two factors 25 .The statistical tools prcomp, ggplot2, and Hmisc from R 4.1.1 were used to conduct principal components, and correlation analyses respectively on the mean data.Falconer and Mackay's method was followed to estimate heritability and genetic advance 26,27 .

Ethics approval and consent to participate
Study protocol complies with relevant institutional, national, and international guidelines and legislation.

Results
The results of the ANOVA suggested significant variations among parents and their F 1 hybrids under salt stress conditions.These differences indicated the existence of genetic diversity in the studied germplasm for salinity tolerance, as shown in (Table 4).Moreover, the treatment and Genotypes × Treatment interaction for all Table 3.The list of the primers.www.nature.com/scientificreports/characteristics were highly significant, indicating that all genotypes responded differently to salt stress (Table 4).
All cotton genotypes experienced a negative impact on their agronomic and yield-related traits under salt stress.The reduction was observed in agronomic traits such as PH, NBP, BW, SCY, lint%, FL, and FS as shown in (Table 5).Interestingly, fiber fineness (FF) was increased under saline environments.Furthermore, the mean values for H 2 O 2 , Na + , CAT, POD, and SOD were increased under salt stress, while the mean values for K + /Na + , K + , TSP, Chla and b were decreased under saline conditions.The GAM was classified into three categories: low (0-10%), moderate (10-20%), and high (20% or higher).In terms of heritability, values exceeding 80% were considered very high, values ranging from 60 to 79% were moderately high, values between 40 and 59% were medium, and values lower than 40% were regarded as low 28 .Under saline conditions, high genetic advance as a percentage of the mean (GAM) was observed for BW, CAT, Chla, Chlb, FF, NBP, POD, SCY, SOD and TSP whereas moderate GAM was observed for PH, LP, Na + , K + , FS, FL, and Car.The heritability estimates for BW, CAT, Car, Chlb, FL, FS, K + , K + /Na + , LP, NBP, PH, POD, SOD, and TSP, were very high whilst moderate heritability estimates were observed for SCY, Na + , H 2 O 2 , FF, and Chla under saline conditions (Table 5).

Biochemical traits
The biochemical traits such as chlorophyll a (Chla), chlorophyll b (Chlb), total soluble proteins (TSP), and carotenoid (CAR) exhibited a statistically significant decrease in all genotypes under salt stress (15 ds/m) (Fig. 3).The application of salt stress treatment led to a more significant reduction in Chla, Chlb, TSP, and CAR levels in  3).Furthermore, certain genotypes and crosses, such as FBS-FALCON × FBG-333, FBS-FALCON × Barani-222, FBS-SMART × FBG-333, and Barani-333 × Barani-222 exhibited less reduction in Chla, Chlb, TSP, and CAR level compared to susceptible genotypes (Fig. 3).The application of NaCl treatment had a significant impact on the ionic homeostasis of plants, particularly on Na + , K + , and the K + /Na + ratio.Notably, the Na + content in NaCl-treated plants was found to be significantly higher compared to the control group, indicating an increase in sodium accumulation as a result of the treatment.The mean graph of (Fig. 4) depicted that certain genotypes and crosses, including FBS-Falcon, Barani-333, JSQ-White Hold, Ghauri, FBS-FALCON × JSQ-White Hold, FBG-222 × FBG-333, FBG-222 × Barani-222, and Barani-333 × FBG-333 lower increase in sodium contents compared other genotypes.Compared to other genotypes, these genotypes also revealed less reduction in potassium contents.Consequently, these genotypes and crosses maintained a higher ratio of potassium to sodium content, similar to that of the control group and

Correlation analysis
The relationship between various morpho-physiological and biochemical traits is crucial in determining the most suitable genotype under both normal and saline environments.The Pearson correlation coefficients were computed separately for normal and saline conditions.Under normal conditions, FL, Na + , TSP, CAT, FS, PH, FF, POD, and SOD showed positive association with each other.Lint % revealed a significant positive relationship with FL, TSP, CAT, FS, FF, POD, Chla, Chlb, BW, and CAR contents under control conditions (Fig. 6).Under salt stress conditions, H 2 O 2 and Na + revealed a significant negative relationship with all morphological, agronomical (SCY, BW, NBP, and LP), fiber quality traits (FL, FS, & FF) and physiological characters (CAT, TSP, SOD, POD, Car, chlorophyll contents, K + /Na + , and K + respectively (Fig. 2).Under salt stress environments all agronomic characters (SCY, BW, NBP, and LP) revealed positive relationship with physiological traits CAT, TSP, POD, Car, chlorophyll contents, K + /Na + , and K + respectively.The fiber quality parameters showed a positive association with all antioxidant and biochemical traits under salt stress conditions (Fig. 7).

Examination of the expression of ERF genes (Ghi-ERF-2D.6 and Ghi-ERF-7A.6) by qPCR
The quantitative measurement of mRNA expression in the leaf of genotypes at salt stress levels of control and 15 ds/m was performed.As an internal control of gene normalization, the GAPDH was employed.In Fig. 10, the genes Ghi-ERF-2D.6 and Ghi-ERF-7A.6 showed relatively higher expression at 15 ds/m.In genotype JSQwhite Hold, the expression of Ghi-ERF-2D.6 is 1.5 folds higher at 15 ds/m than control whereas the expression of Ghi-ERF-7A.6 is 2.2 folds higher at 15 ds/m than control.The genotype Barani-333 × FBG-333 showed 2 folds higher expression of Ghi-ERF-2D.6 at 15 ds/m than control whereas the expression of Ghi-ERF-7A.6 is 2.7 folds higher at 15 ds/m than control.The expression of both Ghi-ERF-2D.6 and Ghi-ERF-7A.6 in genotype FBG-222 × Barani-222 was measured 2 folds higher at 15 ds/m than control.In genotype Ghauri, the expression of Ghi-ERF-2D.6 and Ghi-ERF-7A.6 was 2.5 folds and 2.2 folds higher at 15 ds/m than control respectively.The genotype FBG-222 × FBG-333 showed 1.5 folds and 3 folds higher expression of Ghi-ERF-2D.6 and Ghi-ERF-7A.6 at 15 ds/m than control respectively (Fig. 10).In genotype FBS-FALCON × JSQ-white Gold, the expression of Ghi-ERF-2D.6 is 2 folds higher whereas the expression of Ghi-ERF-7A.6 is 2.7 folds higher at 15 ds/m than control respectively.The genotype Barani-333 showed 2.5 folds and 3 folds higher expression of Ghi-ERF-2D.6 and Ghi-ERF-7A.6 at 15 ds/m than control respectively.The expression of Ghi-ERF-2D.6 and Ghi-ERF-7A.6 in genotype FBS-FALCON was measured 2.1 folds and 2.3 folds higher at 15 ds/m than control respectively.The expression level of both the genes was increased at 15 ds/m and showed significant tolerance against salt stress (Fig. 10).The PCR reaction using short length primers was run to detect the correct size of the gene, which corresponds 120 bp (Fig. 11).

Discussion
Cotton is an economically important crop that is grown worldwide for its fiber and seed oil.However, cotton growth and productivity are often limited by various abiotic stresses, including salt stress 4 .Salt stress can have a significant impact on cotton physiology, leading to reduced growth, disrupted water and ionic balance, oxidative stress, and decrease in yield.Understanding these physiological effects is critical for developing strategies to mitigate the negative effects of salt stress and improve cotton productivity in salt-affected soils 29,30 .
A significant amount of work has been done so far to develop cotton cultivars that can tolerate stress.In this pursuit, plant breeders often depend on the genetic variability present in the available germplasm to select desirable traits 31 .To improve breeding programs aimed at producing salt-tolerant cotton genotypes, it is crucial to have current knowledge on genetic variability and heritability 31 .In this study, Line × Tester approach was used, where four lines and four testers were crossed, resulting in the production of 16 F 1 hybrids.The success of improving crop plants genetically is dependent on the level of heritability of traits that are economically valuable 32 .Traits that exhibit high heritability and genetic advance are more likely to be transmitted to the next generation in greater proportions.When there is both a high h 2 b and high GAM, this can lead to genetic gains through the selection process 33 .In this study, traits such as NBP, BW, LP, SCY, K + /Na + , CAT, SOD, POD, TSP, Car, FL and FS demonstrated moderate to high heritability and GAM, suggesting the presence of additive gene action.These traits can be useful for early-stage selection of genotypes, which can then be utilized in breeding programs focused on improvement 34 .Under salt stress environment, significant reduction was observed in agronomic traits such as PH, NBP, BW, SCY, and lint%.Interestingly, fiber fineness (FF) was increased under saline environments.Furthermore, the mean values for H 2 O 2 , CAT, SOD, and POD, were increased under salt stress, while the mean values for TSP, Car, Chl a and b were decreased under saline conditions 8 .In a stress breeding program, measuring chlorophyll content is crucial in identifying salt-tolerant genotypes, as higher chlorophyll contents correlate with greater salt tolerance.In our study chlorophyll contents were reduced under salt stress conditions 35 .The reduction in chlorophyll contents, leads to increased production of H 2 O 2

18
. This creates oxidative stress due to the strong negative association between H 2 O 2 and chlorophyll content under salt treatment 36 .One of the ways in which oxidative stress can reduce cotton yield under salt stress is by inhibiting photosynthesis.Salt stress-induced oxidative stress can damage the photosynthetic machinery, resulting in a decrease in the efficiency of photosynthesis.This, in turn, reduces the plant's ability to produce energy and fix carbon dioxide, which are essential for growth and yield [37][38][39] .Sodium and potassium are essential ions that play critical roles in different biochemical processes in plants, including osmoregulation, ion homeostasis, and enzyme activation 40 .Salt stress can cause an influx of toxic ions such as sodium and chloride into the plant and decrease the potassium contents in plants, which disrupt ion homeostasis, leading to ion toxicity and osmotic stress 3 .This can impair the plant's ability to maintain normal cellular function, resulting in reduced cotton yield and boll weight.In our study, the genotypes FBS-FALCON × Ghauri-3, FBS-SMART × JSQ-White Hold, FBS-SMART × Barani-222, FBS-SMART × Ghauri-3, FBG-222 × JSQ-White Hold, FBG-222 × Ghauri-3, Barani-333 × JSQ-White Hold, and Barani-333 × Ghauri-3 revealed higher Na + contents and lower values for the K + and K + /Na + and performed poor for agronomic physiological and fiber quality traits.The excess Na + accumulation can also reduce water uptake by the plant, as well as it also competes with other ions, such as potassium and calcium, for binding sites on the root surface and within the plant 8 .This can reduce the uptake of other vital nutrients and lead to nutrient deficiencies, which can further reduce yield of the seed cotton and boll weight 8 .In our study, negative association of    Na + and H 2 O 2 with fiber quality traits and agronomic as well as antioxidant traits was observed under salt stress conditions.It was reported that tolerant plants accumulated a lower amount of Na + ions, while sensitive plants accumulated a higher amount of Na + ions within their cells 41 .Salt-tolerant plants maintain their Na + /K + ratio by reducing the uptake of Na + from the roots, sequestering the excess Na + present in the cytosol into the vacuole, and promoting the efflux of Na + from root cells 41 .
Antioxidants play a critical role in protecting cotton plants from oxidative stress under salt stress.Antioxidants such as catalase, peroxidase, superoxide dismutase and non-enzymatic antioxidants (carotenoids, ascorbate and flavonoids) can neutralize ROS by donating electrons to them, preventing the formation of more ROS and the damage they can cause 4 .In response to salt stress, plants activate protective enzymes such as SOD, POD, and CAT, to enhance their ability to eliminate ROS 6,30 .The current study observed an increase in the activities of SOD, POD, and CAT in leaves under salt stress.The increased SOD activity in leaves may aid in scavenging oxygen-free radicals, while the elevated CAT activity may facilitate the breakdown of H 2 O 2 into water and oxygen, thus reducing its levels.This finding is consistent with previous reports 6,8,10 who also noted an increase in SOD, POD, and CAT activities in cotton leaves under salinity stress.The POD activity was enhanced up to 53% in resistant cultivars 37 .The enhancement of POD activity contributes to the improvement of photosynthetic activity, thereby highlighting the important role of antioxidant defense mechanisms in mitigating salt stress 37 .The enhanced activity of antioxidants such as SOD, POD, and CAT were associated with salt tolerance during fiber development 42 .Under stressful conditions, carotenoids tend to increase as their primary function is to protect against singlet oxygen [43][44][45] .High-yielding cultivars often exhibit elevated levels of CAT, TSP, and POD as they play an active role in regulating H 2 O 2 levels by scavenging it to maintain optimal levels.The genotypes FBS-Falcon, Barani-333, JSQ-White Hold, Ghauri, along with crosses FBS-FALCON × JSQ-White Hold, FBG-222 × FBG-333, FBG-222 × Barani-222, and Barani-333 × FBG-333 revealed higher level of K + /Na + , K + , POD, SOD, CAT, TSP, Chla, Chlb, and CAR and declared as salt tolerant cultivars.The above-mentioned genotypes also showed relatively higher expression level of Ghi-ERF-2D.6 and Ghi-ERF-7A.6 at 15 dS/m and proved the role of ERF genes in salt tolerance in cotton.Similar findings also reported in cotton 46 , maize 47 , quinoa 48 and wheat 49 .

Table 2 .
Analysis of soil properties used in this study.

Catalase (U/mg protein)
22talase activity was determined by measuring the amount of H 2 O 2 consumed and transformed into H 2 O and O 2 .Spectrophotometer values were recorded at 240 nm absorbance to calculate CAT activity22.

Table 4 .
ANOVA split plot design for different agronomic and physiological traits.*Significant at the 0.05 level.**Significant at the 0.01 level.

Table 5 .
Genetic components of variability, genetic advance percentage means and heritability (broad sense) estimates for studied traits across control and salt stress conditions.